Abstract
CRISPR-Cas13 exclusively targets RNA. In prokaryotes, Cas13 cleaves both target and non-target RNA indiscriminately upon activation by a specific target RNA, but in eukaryotic cells collateral cleavage activity has been limited. Here we report that LbuCas13a exhibits strong collateral RNA cleavage activity in human cells when delivered as ribonucleoprotein, independent of cell line and targeting both exogenous and endogenous transcripts. Collateral RNA cleavage starts within 50 minutes of ribonucleoprotein delivery resulting in major alterations to the total RNA profile. In response to the collateral RNA cleavage, cells upregulate genes associated with the stress and innate immune response, ultimately leading to apoptotic cell death. This enables us to use LbuCas13a as a flexible and repeatable target-RNA-specific cell elimination tool. Finally, using both total RNA sequencing and Nanopore sequencing, we find that LbuCas13a activation leads to rapid and near-global depletion of cytoplasmic RNAs, and that cleavage occurs at specific nucleotide positions.